Magnetic microbeads are used to enrich cells, proteins, and nucleic acids from complex samples using biomagnetic separation. The process requires a well-designed magnetic separation rack, and the beads need to be coated and functionalized in order to capture the desired target molecule. Magnetic microbeads are typically made of iron oxide (Fe3O4) also known as magnetite, and are 0.5 to 500 μm in diameter. The diameter of the microbeads is a function of the non-ferrous material composing the beads.(more information below). The microbeads are small enough that they are superparamagnetic, which means that they are inherently non-magnetic, but they become magnetized when placed into a magnetic field. This effect is reversible, and the magnetism disappears again after the microbeads are removed from the magnetic field.
A sonicator bath is a tool that propagates ultrasonic waves through fluid contained within it. The ultrasonic bath is used in the laboratory to lyse cells, to degass water, and to break up clumped and aggregated magnetic beads, among many other uses. Ultrasonic cleaners are used to remove dirt and grime on objects that are hidden in difficult crevices that brushes or sprays cannot access. The most common fluid used in an ultrasonic bath or ultrasonic cleaner is distilled water. Other solvents may be added to help in cleaning processes, but in the laboratory, sonicator baths are almost always filled with distilled water. One must be careful when using solvents to ensure that they don’t have a low flash point as the ultrasonic waves will heat up the fluid in the bath. Sonicator baths work by applying ultrasonic waves to fluid. Ultrasonic waves are sound waves greater than 20 kHz; when propagated through fluid they bounce into air bubbles and cause them to burst. The shock wave released by bursting air bubbles helps to lyse cells, remove dirt from surfaces, or to break apart aggregated magnetic beads.
Recrystallization is the process of obtaining pure crystals of a compound from a solution containing impurities. Hot gravity filtration is commonly used to remove these impurities from a solution prior to recrystallization. Hot filtration is necessary for recrystallization when impurities exist in solution. Firstly, recrystallization requires a hot solution because the solution needs to be super saturation in order for crystals to form upon cooling. Secondly, the impurity may have different solubility in certain solvents than the compound to be crystallized. The idea is to choose a solvent that dissolves the compound to be crystallized upon heating, but the impurity doesn’t dissolve in the solvent at high temperatures. The impurity is then filtered out during the hot gravity filtration process.
The BCA assay is used to quantify protein concentration by using bicinchoninic acid to identify copper ions reduced by protein in a biuret reaction. The BCA protocol requires a working solution mixed with the sample; when protein is present, the reaction produces a purple color that absorbs light at 562 nm and is quantified with a spectrophotometer. The BCA assay is similar to other protein quantification assays such as Lowry or Bradford assays. However, the biuret reaction of the BCA assay occurs between the nitrogens on the peptide backbone and copper as well as nitrogens on the amino acid side chains. The fact that the peptide backbone participates in the reaction means that the BCA assay is more consistent between proteins and is less dependent upon amino acid composition. The BCA protocol is simple and quick. If the sample is heated to 37°C, then the incubation time is only 30 minutes, and the absorbance measurement takes only a few minutes. The BCA assay is an excellent method for quantifying total protein concentration after biomagnetic protein purification.
Multiplex immunoassays enable the detection of multiple different analytes in the same sample. A well-designed multiplex assay could mean that only one vial of blood might need to be drawn from a patient instead of 8 or more vials. Therefore, one multiplex test can answer multiple questions at the same time. Immunoassays capitalize upon the specific affinity between antibody and antigen. Some immunoassays use antigen as the probe in order to detect the binding of antibody target, while other immunoassays use antibody probes in order to detect antigen targets. Most immunoassays are single-plex, meaning that they can only be used to detect one antibody-antigen pair at a time; this is generally the case with most ELISA (Enzyme Linked ImmunoSorbent Assay) tests. The idea of a multiplex ELISA is attractive, and it mostly indicates a type of multiplex immunoassay that relies upon antibody-antigen binding events. The traditional ELISA is single-plex, and requires multiple binding and washing steps as well as an enzymatic system that produces a colorimetric or chemiluminiscent label as a quantitative readout of target concentration in a sample.
Sonication of cells is an essential first step to any protein purification process. Sonication is used to break apart the cell membrane, which releases all proteins into solution. Once the intracellular and transmembrane proteins are free, they can be enriched by protein purification methods. One very useful method is biomagnetic protein purification. This process uses superparamagnetic beads to isolate specific target proteins. The superparamagnetic beads are coated with proteins that specifically bind to the proteins of interest, and a magnetic separation rack attracts the beads. The remaining cellular debris is washed away and replaced by a protein isolation buffer that keeps the proteins stable until further analysis can be performed.
Cellular assays form the backbone of basic research and drug discovery. A cell culture is the perfect environment in which to collect information about normal and abnormal growth, and to test novel drug compounds safely in a controlled environment. An assay in biology is carefully designed to test a single variable. Once a standard protocol for a cellular assay is established, it is highly consistent, so the chances of having confounding variables is low. This streamlines experiments and makes it easy to analyze data and draw conclusions.
A magneto elisa is a combination of magnetic bead separation and Enzyme Linked ImmunoSorbent Assay (ELISA) for analyte detection. The magnetic bead separation helps to enrich the target population from complex media such as serum or whole blood prior to quantitative detection via ELISA. This works particularly well for cell separation and detection. One example where a magneto ELISA was used, was to detect CD4+ T-cells from whole blood of HIV patients. An accurate count of CD4+ T-cells is imperative in the treatment and management of HIV and detection of AIDS development.
Proteomics is the study of the protein in an organism. Protein is a fundamental building block of life, and proteins are the workhorses within and between cells. Biochemical pathways are built out of enzymes and ligands—without them nothing would be accomplished; plants wouldn’t produce glucose, animals wouldn’t be able to digest food, the immune system would cease to exist, and all other biological processes would grind to a halt. The fundamental importance of proteins for life makes them an important topic of study. The first step in understanding protein structure and function is to extract them. Protein extraction is the process of isolating and purifying protein from samples of whole tissue, cell cultures, or biological fluids. The protein extraction protocol used is tailored to match the starting material and the end goals of the assay. Considering the goal of the experiment is extremely important when developing a protein extraction protocol because certain buffer choices (such as high salt, high detergent formulations) can ruin an experiment when higher order protein structure and function needs to be preserved.
Cell lysis is the first step of breaking the cell membrane that enables further study of the proteins, nucleic acids, and other molecules inside of cells. When cell lysis is successful, the undamaged contents of the cell escape through the damaged cell membrane. These contents are then separated out of the mixed sample and used for further study. The methods used for separation of the lysed cell contents are dependent on the goal of the study. Careful investigation of these inner workings can reveal disease patterns, improve our understanding of normal cellular function, and elucidate biochemical pathways and therapeutic targets. Protein isolation is different from nucleic acid separation, and the reagents used vary drastically. There are a few ways to lyse the cell membrane; these include mechanical disruption, liquid homogenization, freeze/thaw cycles, manual griding, and the use of detergents. Sonication cell lysis is an example of mechanical disruption used for releasing the contents of cells.